Platelet Aggregation Inhibitory Activity Research Articles

Analysis and Identification of Putative Novel Peptides Purified from Iranian Endemic Echis Carinatus Sochureki Snake Venom by MALDI-TOF Mass Spectrometry.

The Iranian Echis Carinatus (IEC) venom is an exclusive natural source of bio-substances for a wide range of purposes in the blood coagulation cascade. The present study for the first time was aimed to assess novel pro-coagulant, anti-coagulant and anti-platelet proteins, named EC1.5 (a), EC5.1 (b) and EC4 (a) from Iranian Echis Carinatus (IEC) venom. These peptides were purified by multi-step chromatography methods. Hematological properties were measured using activated clotting tests, platelet aggregation studies, and hemorrhage assessment. Subsequently, these proteins were identified through both their intact molecular mass and peptide mass fingerprint (PMF) using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Multiple sequence alignments were performed by ClustalW, Bioedit software. Molegro Data Modeller (MDM) 3.0 software was used to predict the putative tertiary structure of proteins.EC1.5 (a), a single-band protein with a molecular mass of 66 and 55 kDa, was observed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis as a reduced and non-reduced state, respectively. Based on the Mascot results, we considered that EC1.5 (a) is a metalloproteinase of group ΙΙ which exhibited potent pro-coagulant activity. It is predicted that the EC1.5 (a) with hemorrhagic activity, potentially is a metalloproteinase/disintegrin region that constitutes the disintegrin-like domains. Our findings demonstrate that the disintegrin domain of EC1.5 (a) lacks platelet aggregation inhibitory activity. On the contrary, this factor shows the property of a platelet aggregation inducer. Also, the EC5.1 (b) was observed as a single-band protein with a molecular mass of 7.5 kDa. EC5.1 (b) showed both anti-coagulant and anti-platelet properties. Additionally, the structure of the EC5.1 (b) fraction is expected to be similar to that of phospholipase A2, while EC4 (a) structure is potentially very similar to that of Echistatin with 5 kDa molecular mass. We introduce the predicted structure of P-II snake venom metalloproteinase/ disintegrin domains, phospholipase A2 and Echistatin-like fractions. Further research is therefore needed to determine the complete structure of these novel fractions and elucidate their mechanism of action and future therapeutic applications of cardiovascular and homeostasis disorders.

Antiplatelet Effects of Selected Xanthine-Based Adenosine A2A and A2B Receptor Antagonists Determined in Rat Blood.

The platelet aggregation inhibitory activity of selected xanthine-based adenosine A2A and A2B receptor antagonists was investigated, and attempts were made to explain the observed effects. The selective A2B receptor antagonist PSB-603 and the A2A receptor antagonist TB-42 inhibited platelet aggregation induced by collagen or ADP. In addition to adenosine receptor blockade, the compounds were found to act as moderately potent non-selective inhibitors of phosphodiesterases (PDEs). TB-42 showed the highest inhibitory activity against PDE3A along with moderate activity against PDE2A and PDE5A. The antiplatelet activity of PSB-603 and TB-42 may be due to inhibition of PDEs, which induces an increase in cAMP and/or cGMP concentrations in platelets. The xanthine-based adenosine receptor antagonists were found to be non-cytotoxic for platelets. Some of the compounds showed anti-oxidative properties reducing lipid peroxidation. These results may provide a basis for the future development of multi-target xanthine derivatives for the treatment of inflammation and atherosclerosis and the prevention of heart infarction and stroke.

Analysis and Identification of Putative Novel Peptides Purified from Iranian Endemic Echis Carinatus Sochureki Snake Venom by MALDI-TOF Mass Spectrometry

The Iranian Echis Carinatus (IEC) venom is an exclusive natural source of bio-substances for a wide range of purposes in the blood coagulation cascade. The present study for the first time was aimed to assess novel pro-coagulant, anti-coagulant and anti-platelet proteins, named EC1.5 (a), EC5.1 (b) and EC4 (a) from Iranian Echis Carinatus (IEC) venom.These peptides were purified by multi-step chromatography methods. Hematological properties were measured using activated clotting tests, platelet aggregation studies, and hemorrhage assessment. Subsequently, these proteins were identified through both their intact molecular mass and peptide mass fingerprint (PMF) using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Multiple sequence alignments were performed by ClustalW, Bioedit software. Molegro Data Modeller (MDM) 3.0 software was used to predict the putative tertiary structure of proteins.EC1.5 (a), a single-band protein with a molecular mass of 66 and 55 kDa, was observed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis as a reduced and non-reduced state, respectively. Based on the Mascot results, we considered that EC1.5 (a) is a metalloproteinase of group ΙΙ which exhibited potent pro-coagulant activity. It is predicted that the EC1.5 (a) with hemorrhagic activity, potentially is a metalloproteinase/disintegrin region that constitutes the disintegrin-like domains. Our findings demonstrate that the disintegrin domain of EC1.5 (a) lacks platelet aggregation inhibitory activity. On the contrary, this factor shows the property of a platelet aggregation inducer. Also, the EC5.1 (b) was observed as a single-band protein with a molecular mass of 7.5 kDa. EC5.1 (b) showed both anti-coagulant and anti-platelet properties. Additionally, the structure of the EC5.1 (b) fraction is expected to be similar to that of phospholipase A2, while EC4 (a) structure is potentially very similar to that of Echistatin with 5 kDa molecular mass. We introduce the predicted structure of P-II snake venom metalloproteinase/ disintegrin domains, phospholipase A2 and Echistatin-like fractions. Further research is therefore needed to determine the complete structure of these novel fractions and elucidate their mechanism of action and future therapeutic applications of cardiovascular and homeostasis disorders.

Pharmacodynamic and Pharmacokinetic Drug Interactions between Fexuprazan, a Novel Potassium-Competitive Inhibitor, and Aspirin, in Healthy Subjects

Acid-reducing agents are commonly used for the prevention of aspirin-induced gastrointestinal complications such as peptic ulcers. As a novel potassium-competitive acid blocker, fexuprazan is expected to prevent aspirin-induced gastrointestinal complications. This randomized, open-label study aimed to evaluate the pharmacodynamic and pharmacokinetic interactions between aspirin and fexuprazan in healthy Koreans. Subjects randomized to the aspirin group received 500 mg aspirin in combination with 80 mg fexuprazan. For the fexuprazan group, fexuprazan 80 mg was administered alone and then in combination with aspirin 500 mg. Platelet aggregation inhibited by aspirin and the pharmacokinetic parameters of aspirin and fexuprazan were compared between monotherapy and combination therapy. A total of 22 subjects completed the study. The platelet aggregation-inhibitory activity and systemic exposure to aspirin were not significantly affected by fexuprazan coadministration. The systemic exposure of fexuprazan was decreased up to 20% by aspirin coadministration, which was not regarded as clinically meaningful considering the previously reported exposure-response relationship. In conclusion, there were no clinically relevant pharmacodynamic or pharmacokinetic interactions between aspirin and fexuprazan. This finding suggests the potential of fexuprazan for the prevention of aspirin-induced gastrointestinal complications, serving as a baseline for optimizing its therapeutic application with aspirin.

CENTIPEDE VENOM TOXINS AND ITS BIOMEDICAL AND PHARMACOLOGICAL PROPERTIES

The present review article explains venoms from various centipede species with their biomedical and pharmacological properties. Centipede venom is a natural source of bioactive proteins, peptides and other small molecules. These use venoms as defense arsenals to paralyze prey. This review paper sketch out important physiological effects like platelet aggregation, anticoagulant, phospholipase A2 and trypsin inhibiting activity. Centipede venom toxins selectively bind Kv2.1 channel and block them. Centipede venom disrupts cardiovascular, respiratory, muscular and nervous systems by targeting the broadly distributed KCNQ channels. It also signifies toxin-voltage-gated integrations and its inhibition. These peptides can be used for developing drugs for treatments as well as bio-insecticides for insect control.

A Comprehensive Review on Therapeutic Potentials of Natural Cyclic Peptides

Abstract: Cyclic peptides have emerged as a promising class of organic compounds that possess polypeptide chains with a cyclic ring structure. There is a circular sequence of bonds in which the ring structure is formed via linkage between one end of the peptide bond and the other end with an amide bond or any other chemically stable bonds like ether, thioether, lactone, and disulfide. Generally, the cyclic peptides are isolated from natural resources like invertebrate animals, micro-organisms of marine habitats, and higher plants. These cyclic peptides possess unique structures with diverse pharmacological activities. Nowadays, cyclic peptides possess superior therapeutic value due to several reasons such as greater resistance to enzymatic degradation (in vivo) and higher bio-availability. Some of these cyclic peptides are rich in leucine, proline, while some have other amino acids as their major constituents. Numerous novel cyclic peptides isolated from natural sources are successfully developed as bioactive products. Recently, cyclic peptides derived from natural resources have attracted attention for exploring their numerous beneficial effects. Moreover, it is reported that natural cyclic peptides exhibit various therapeutic activities like an anthelmintic, ACE inhibitor, anti-tumor, microtubule inhibitor, anti-fungal, antimalarial, and platelet aggregation inhibiting activity. In this review, various cyclic peptides are reported with structures and biological activities that are isolated from various natural sources. The natural cyclic peptides possess a wide spectrum of biological activities and can become a drug of the future for replacing the existing drugs which develop resistance.

Hawanoids A‒E, unprecedented diterpenoids with PAF-induced platelet aggregation inhibitory activities from the deep-sea-derived fungus Paraconiothyrium hawaiiense

Hawanoids A‒E (1‒5), five highly cyclized diterpenoids were isolated from the deep-sea-derived fungus Paraconiothyrium hawaiiense FS482. Compounds 1 and 2 possessed an unprecedented tetracyclo[6.6.2.02,7.011,15]cetane carbon skeleton while 3 and 4 possessed an unusual 11,14-macrocyclic ether moiety in phomactin family. Their structures including the stereo-chemistry were determined through spectroscopic analysis, X-ray diffractions and computational calculations. The plausible biosynthetic pathway was proposed based on the predicted biosynthetic gene cluster. All of the isolated compounds exhibited inhibitory activities against PAF-induced platelet aggregation. The molecular docking study was carried out understand the interaction between the PAF receptor and hawanoids with different skeletons.

Discovery of Natural Product Inspired 3-Phenyl-1H-isochromen-1-ones as Highly Potent Antioxidant and Antiplatelet Agents: Design, Synthesis, Biological Evaluation, SAR and In Silico Studies.

Several natural/synthetic molecules having a structure similar to 1H-isochromen- 1-ones have been reported to display promising antioxidants and platelet aggregation inhibitory activity. Isocoumarin (1H-2-benzopyran-1-one) skeleton, either whole or as a part of the molecular framework, has been explored for its antioxidant or antiplatelet activities. Based on the literature, a new prototype, i.e., 3-phenyl-1H-isochromen-1-ones based compounds, has been rationalized to possess both antioxidant as well as antiplatelet activities. Consequently, no reports are available regarding its inhibition either by cyclooxygenase-1 (COX-1) enzyme or by arachidonic acid (AA)-induced platelet aggregation. This prompted us to investigate 3-phenyl-1H-isochromen-1-ones towards antioxidant and antiplatelet agents. The goal of this work was to identify new 3-phenyl-1H-isochromen-1-ones based compounds via synthesis of a series of analogues, followed by performing in vitro antioxidant as well as AA-induced antiplatelet activities. Then, identification of potent compounds by SAR and molecular docking studies was carried out. Out of all synthesized 3-phenyl-1H-isochromen-1-ones analogues, five compounds showed 7-fold to 16-fold more highly potent antioxidant activities than ascorbic acid. Altogether, ten 3-phenyl-1H-isochromen- 1-one analogues displayed antioxidant activities in 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay. Almost all the 3-phenyl-1H-isochromen-1-one analogues exhibited potent AA-induced antiplatelet activity; few of them displayed 7-folds more activity as compared to aspirin. Further, in silico analysis validated the wet results. We disclose the first detailed study for the identification of 3-phenyl-1H-isochromen-1-one analogues as highly potent antioxidant as well as antiplatelet agents. The article describes the scaffold designing, synthesis, bioevaluation, structure-activity relationship, and in silico studies of a pharmaceutically privileged bioactive 3-phenyl-1H-isochromen-1-one class of heterocycles.

Amide Derivatives of Ginkgolide B and Their Inhibitory Effects on PAF-Induced Platelet Aggregation.

Ginkgolides are the most important components of Ginkgo biloba extracts, whose lactone can be hydrolyzed in the aqueous environment. Although the hydrolyzed products have complex structures and their functions are not well-understood, opening the lactone ring is an important strategy in producing novel derivatives of ginkgolide. The preparation of a single pure aminolyzed ginkgolide for the study of its bioactivity and understanding of the process of aminolysis are challenging. To obtain stable aminolyzed products, four amide derivatives (2–5) of ginkgolide B (GB, 1) were prepared via the ring-opening reaction of its lactone with propylamine. These products were purified and fully identified by high-resolution mass spectrometry (HRMS) and nuclear magnetic resonance (NMR) spectroscopy and were further evaluated for their ability to inhibit the PAF-induced platelet aggregation of rabbit platelets in vitro. Compound 2, which was obtained by selective aminolysis of the lactone ring C of GB, showed a much better inhibitory activity of platelet aggregation (IC50, 15 nM) than the parent compound GB (IC50, 442 nM). The other three products (3–5), which were obtained by the aminolysis of lactone rings C and F of GB, did not show platelet aggregation inhibitory activity. The results greatly extended our understanding of the chemistry of GB and provided important structural information for the exploration and development of new drugs based on ginkgolides in G. biloba.

Macrostachyols A-D, oligostilbenes from Gnetum macrostachyum inhibited in vitro human platelet aggregation

Introduction: Gnetum macrostachyum is a known Thai medicinal plant as a source of bioactive oligostilbenes, which possess platelet inhibitory activities. The study aimed to evaluate the in vitro human platelet aggregation inhibitory activities of macrostachyols A-D (compounds 1-4) isolated from the roots of G. macrostachyum. Methods: The in vitro human platelet aggregation assay was assayed with a 96-well microtiter plate format. The well-known aggregating agents were used to investigate the possible mechanism of inhibition, including adenosine diphosphate (ADP), arachidonic acid (AA), thromboxane A2 analog (U-46619), collagen, thrombin, and thrombin receptor-activating peptide-6 (TRAP-6). Results: Compound 1 was more potent than ibuprofen (positive control) on the adenosine diphosphate- induced platelet aggregation assay (P < 0.05). Compound 3 was more potent than 1, 2, and 4 (P < 0.05), but all active oligostilbenes were less potent than the positive control (P < 0.05) on the arachidonic acid-induced platelet aggregation assay. The oligostilbenes 1, 2, 3, and 4 also displayed the inhibitory effects on the U-46619-induced platelet aggregation. The tetrameric stilbenes 1 was the only compound that exhibited inhibitory effects on thrombin-induced platelet aggregation without TRAP-6 mediated platelet aggregation. Conclusion: The findings revealed the inhibitory effects of oligostilbenes on human platelet aggregation through a target-specific experimental design. It suggests that oligostilbenes from this plant might be applied as antiplatelet aggregation agents in platelet hyperreactivity- related diseases.

A Tangy Twist Review on Hog-Plum: <i>Spondias pinnata</i> (L.f.) Kurz

<em>Spondias pinnata</em> (L.f.) Kurz., belongs to the Anacardiaceae family, generally referred to as Indian Hog plum, a deciduous, glabrous tree with a healthy fruit to eat. Hog plum is a mild deciduous tree plant of high nutritional value, stubby calories and abundant in vegetable proteins, zinc, chitin, starch, vitamins and minerals. Traditional use of various parts of <em>S. pinnata</em> includes diarrhea, dysentery, stomach troubles, hyperacidity, wounds, sprain, rheumatism, gonorrhoea, tuberculosis, aphrodisiac, arrow poison antidote, dyspepsia, dysentery, ring worms, abscess painful joints, refrigerant, tonic, antiseptic, astringent, mental disorders, tuberculosis, vomiting and many more. A number of pharmacological activities were reported from several extracts of <em>S. pinnata</em> and its parts that include hypoglycemic, anti-cancer, ulcer-protective, antidiarrhoeal, anti-microbial, hepatoprotective, thrombolytic, anti-inflammatory, antiarthritic, analgesic, antipyretic, antihypertensive, anthelmintic, diuretic and laxative, anti-tuberculosis, cytotoxic, antioxidant, anti-hyperlipidemic, ischemia reperfusion injury and preconditioning of heart, anxiolytic, reduce side effects of chemotherapy, ameliorating, platelet aggregation inhibitory activity and acute and sub-chronic toxicity. A few phytochemicals were detailed on this plant. The chemical components of <em>S. pinnata</em> include different amino acids, carbohydrates, terpenoids, flavonoids, polysaccharides, steroids and so on. Various solvent extract and their gas chromatography-mass spectrometry analysis have confirmed the structures of a number of important phytoconstituents. Hence this review can be a good reference for researchers who would undertake further investigation about <em>S. pinnata</em>.

Screening for the antiplatelet aggregation quality markers of Salvia yunnanensis based on an integrated approach

This study aimed to identify the antiplatelet aggregation quality markers of Salvia yunnanensis (SY) based on an integrated approach. The effects of SY methanol extracts on platelet aggregation were measured to evaluate their in vitro biological activity. Chemical composition differences were determined by HPLC. Molecular docking methods were used to assess the action mechanism of the potential active compounds and target proteins in SY. The results showed that 12 batches of SY samples inhibited platelet aggregation. Sodium danshensu, protocatechuic aldehyde, rutin, rosmarinic acid, salvianolic acid B, dihydrotanshinone I, tanshinone I, cryptotanshinone, and tanshinone IIA components were identified using chemical fingerprints. Multivariate statistical analysis indicated that cryptotanshinone, dihydrotanshinone I, tanshinone I, tanshinone IIA, and rosmarinic acid may have been the active components for antiplatelet aggregation, and molecular docking showed that these five components could combine with P2Y12 protein on platelets. Furthermore, platelet aggregation inhibition activity of the five monomers was verified separately and it was obvious that cryptotanshinone and rosmarinic acid inhibited platelet aggregation. Therefore, cryptotanshinone and rosmarinic acid may be the quality markers of SY. This report describes a comprehensive, scientific, and feasible method for SY quality evaluation and provides a preliminary scientific basis for applying the antithrombotic activity of SY.

Pharmacological Modulation of Smooth Muscles and Platelet Aggregation by Psidium cattleyanum.

Traditionally, in the Southern Asian countries, Psidium cattleyanum is a widely used plant for the management of various ailments such as gastrointestinal, respiratory, and cardiac disorders, but it lacks proof on a scientific basis, and therefore, this is the major emphasis of the current research work. Crude extract of Psidium cattleyanum (Pc.Cr) was preliminary analyzed for the presence of different classes of bioactive molecules. The aqueous and dichloromethane fractions of Pc.Cr were subjected to in vitro and in vivo studies. It was applied at variable concentrations (0.1–10 mg/ml) to isolated rabbit jejunum to investigate spasmolytic effect. Concentration dependent curves of calcium were constructed to check the calcium channel antagonistic activity. For the evaluation of tracheorelaxant activity, isolated tracheal tissue was treated with High-K+ (80 mM) and carbachol (CCh) and then challenged cumulatively with Pc.Cr. To study the antidiarrheal effect of the plant extract, castor oil-induced diarrhea model was adopted. For evaluation of the hypotensive effect of Pc.Cr, it was given intravenously to preanesthetized normotensive rats, and the response was recorded using pressure transducer. Platelet rich plasma was used for the assessment of the antiplatelet activity when challenged with purinergic and adrenergic agonists. Concentration-dependent inhibition of spontaneous and High-K+ mediated contractions in isolated jejunum was observed by the application of Pc.Cr. Contractions induced in isolated tracheal tissue by High-K+ and CCh were inhibited by application of Pc.Cr to these tissues. Similarly, application of Pc.Cr to High-K+ and phenylephrine (PE) treated aortic strips resulted in vasodilation. Platelet aggregation inhibition was shown by Pc.Cr against adenosine diphosphate (ADP) only. The antidiarrheal effect was observed as a reduction in the total number of feces in Pc.Cr-treated mice when given castor oil. Dose-dependent hypotension was seen in normotensive rats when treated with Pc.Cr intravenously. This study showed the spasmolytic, tracheorelaxant, vasodilator, platelet aggregation inhibitory, antidiarrheal, and hypotensive activities of P. cattleyanum which may be due to the blockage of calcium channels, but the involvement of any other pathway cannot be ignored.

In vitro antiplatelet activity tests conducted on bromelain extracted from the pineapple plant and purified by ion-exchange chromatography using a diethylaminoethyl cellulose xolumn

The present study is aimed at isolating and purifying the bromelain enzyme extracted from the cores of the fruit of the pineapple plant (Ananas comusus). The process of purification of the crude enzyme extract included, in sequence, fractionation through ammonium sulfate precipitation, dialysis, and ion-exchange chromatography. Fractionation of the crude enzyme extract with ammonium sulfate resulted in an increase of the specific activity. The fraction with the highest specific activity was that obtained by precipitation with 50–80 % ammonium sulfate. For this fraction, the specific activity was 8.683 U/mg and the degree of purity of the enzyme was 160 times greater than that of the crude enzyme extract. Further purification by column chromatography using diethylaminoethyl cellulose (DEAE-C) as ion-exchange medium produced six fractions (out of 92) characterized by peaks in proteolytic activity. In particular, the highest proteolytic activity (10.530 U/mg) was displayed by the fifth such fraction. The platelet aggregation inhibitory activity of the DEAE-C-purified bromelian extract was assayed through the Born’s method that uses platelet-rich plasma and asetosal as positive control. The aggregation percent of this bromelain extract was measured to be 49.70% and its inhibition percent 46.89 %.

Structure Activity Relationship of 4-Amino-2-thiopyrimidine Derivatives as Platelet Aggregation Inhibitors

Platelet aggregation plays a pathogenic role in the development of arterial thrombi, which are responsible for common diseases caused by thrombotic arterial occlusion, such as myocardial infarction and stroke. Much efforts are directed toward developing platelet aggregation inhibitors that act through several mechanisms: The main antiplatelet family of COXinhibitors, phosphodiesterase inhibitors, and thrombin inhibitors. Recently, the important role in the platelet aggregation of adenosine diphosphate (ADP)-activated P2Y12 and P2Y1 receptors, Gprotein coupled receptors of the P2 purinergic family, has emerged, and their inhibitors are explored as potential therapeutic antithrombotics. P2Y12 inhibitors, i.e. clopidogrel, prasugrel, ticagrelor, and cangrelor, are already used clinically to reduce coronary artery thrombosis risk and prevent acute coronary syndromes. The search for new P2Y12 inhibitors, with better risk-to-benefit profiles is still ongoing. Several years ago, our group prepared a series of 6-amino-2-thio-3H-pyrimidin-4-one derivatives that displayed an interesting platelet aggregation inhibiting activity. In order to probe the structure-activity relationships and improve their inhibitory effects of these compounds, we synthesized variously substituted 6-amino-2-thio-3H-pyrimidin-4-one derivatives and substituted 4-amino-2-thiopyrimidine-5-carboxylic acid analogues. All the synthesized compounds were tested by light trasmission aggregometry (LTA) as inducers or inhibitors of platelet aggregation in citrated platelet-rich plasma (PRP). Among the 6-amino-2-thio-3H-pyrimidin-4-one derivatives, compounds 2c and 2h displayed marked inhibitory activity, with a capability to inhibit the ADP(10-6M)-induced platelet aggregation by 91% and 87% at 10-4M concentration, respectively. Selected 4-amino-2- thiopyrimidine-5-carboxylic acid derivatives were tested as P2Y12 and P2Y1 antagonists and found to display negligible activity. These negative findings demonstrated that this heterocyclic nucleus is not a useful common pharmacophore for developing P2Y-dependent inhibitors of platelet aggregation. Nevertheless, compounds 2c and 2h could represent a new chemotype to further develop inhibitors of platelet aggregation.

Inhibition of thrombin, an unexplored function of retinoic acid

Retinoic acid, a derivative of vitamin A, is known to possess in vivo anti-inflammatory, anti-platelet and fibrinolytic activities. We have investigated the in vitro thrombin and platelet aggregation inhibitory activities of vitamin A (retinol) and its derivatives, retinoic acid and retinaldehyde. The thrombin enzymatic assay was performed fluorimetrically to assess the inhibition of thrombin (Sigma and plasma). Retinoic acid, retinaldehyde and retinol exhibited potent inhibition of thrombin, with IC50 values of 67μg/ml, 74μg/ml and 152μg/ml, respectively for the inhibition of thrombin (Sigma); and 49μg/ml, 74μg/ml and 178μg/ml, respectively for the inhibition of thrombin (plasma). Amongst vitamin A and its derivatives, retinoic acid showed the highest inhibition of both the forms of thrombin. Vitamin A and its derivatives also displayed remarkable inhibition of platelet aggregation. This is the first report of vitamin A and its derivatives showing inhibition of thrombin and platelet aggregation in vitro.

Pharmacological Studies Pertaining to Smooth Muscle Relaxant, Platelet Aggregation Inhibitory and Hypotensive Effects of Ailanthus altissima.

Objective This in vitro and in vivo study was conducted to rationalize some of traditional medicinal uses of Ailanthus altissima in gastrointestinal, respiratory, and cardiovascular systems. Materials Crude extract of Ailanthus altissima (Aa.Cr) and its fractions were prepared and utilized in in vitro and in vivo studies. For in vitro studies, Aa.Cr was investigated on isolated rabbit jejunum, isolated tracheal strip, and isolated aorta of rat suspended in tissue organ bath. Platelet rich and platelet poor plasma were used to study platelet aggregation inhibitory activity. In vivo antidiarrheal effect of Aa.Cr was investigated on balb/c mice pretreated with castor oil to induce diarrhea and SD rats were used to study hypotensive activity. Results Concentration dependent spasmolytic effects of Aa.Cr and its DCM fraction (Aa.DCM) were observed on spontaneous and spasmogen induced contractions in jejunum isolated from rabbit, but effect against high potassium (high-K+) induced contractions was more potent. Moreover Aa.Cr showed parallel shifting of calcium response curve to the right side. While its aqueous fraction (Aa.aq) caused spasmogenesis of isolated rabbit jejunum, this effect was blocked partially with prior administration of atropine (1μM). Concentration dependent protection against castor oil induced diarrhea was also observed. Relaxant effect was observed by the application of Aa.Cr and Aa.DCM against high-K+ and carbachol (CCh) induced contractions in tracheal strips isolated from SD rats, while Aa.Aq caused partial relaxation of high-K+ induced contractions, but no effect was observed against CCh induced contractions. Relaxation of rat aorta by the application of Aa.Cr and its fractions was also observed. Inhibition of force of contraction in rabbit atrium was also observed. Inhibition of platelet aggregation was observed against epinephrine and ADP induced aggregation. Conclusion Keeping in view the observed results, it is concluded that smooth muscle relaxant, platelet aggregation inhibitory and hypotensive effect may be due to the blockage of calcium channels.

Synthesis of Novel 3(N,N-dialkylamino)alkyl/phenyl Substituted Thieno [2,3-d]pyrimidinones as H1-Anti-Histaminic and Antimicrobial Agents

Background: Thienopyrimidines are the bioisoster of quinazoline and unlike quinazoline exist in three isomeric forms corresponding to the three possible types annulation of thiophene to the pyrimidine ring viz thieno[2,3-d] pyrimidine, thieno[3,2-d] pyrimidine and thieno[3,4-d]pyrimidine. Heterocyclic containing the thienopyrimidinone moiety exhibits various pronounced activities such as anti-hypertensive, analgesic and anti-inflammatory, antiviral, platelet aggregation inhibitory, antiprotozoal bronchodilatory, phosphodiesterase inhibitory, antihistaminic, antipsychotic and antimicrobial activity. Objective: Synthesis of novel 3(N,N-dialkylamino)alkyl/phenyl substituted thieno[2,3-d]pyrimidinones as H1-anti-histaminic and antimicrobial agents. Methods: A series of 3-[(N,N-dialkylamino)alkyl/phenyl]-2-(1H)thioxo-5,6,7,8-tetrahydrobenzo(b) thieno(2,3-d)pyrimidine-4(3H)-ones[4a-d], their oxo analogous [5a-d] and 3-[(N,N-dialkylamino)alkyl]- 2-chlorophenyl-5,6,7,8-tetrahydrobenzo(b)thieno(2,3-d)pyrimidine- 4 (3H)-ones[6a-d]derivative were synthesized from 2-amino-4,5,6,7-tetrahydrobenzo(b)thiophene-3-carboxylic acid by nucleophilic substitution of different N,N-dialkyl alkylene/phenylene diamines on activated 3-acylchloride moiety followed by cyclocondensation with carbon disulfide and ethanolic potassium hydroxide to get [4a-d] and in second reaction by condensation with 4-chlorobenzoyl chloride to get [6a-d] by single pot novel innovative route. The oxo analogous [5a-d] were prepared by treating derivatives [4a-d] with potassium permagnate in ethanolic KOH. The synthesized compound were evaluated for H1-antihistaminic and antimicrobial activities. Results: All synthesized compounds exhibited significant H1-antihistaminic activity by in vitro and in vivo screening methods and data were verified analytically and statistically. The compound 4a, 4b, 5a and 5b showed significant H1-antihistaminiic activity than the reference standard chlorpheniramine maleate. The compound 6d, 6c, 5c and 4c exhibited significant antimicrobial activity.

Discovery of Aporphine Analogues as Potential Antiplatelet and Antioxidant Agents: Design, Synthesis, Structure-Activity Relationships, Biological Evaluations, and in silico Molecular Docking Studies.

To explore the potential of aporphine alkaloids, a novel series of functionalized aporphine analogues with alkoxy (OCH3 , OC2 H5 , OC3 H7 ) functional groups at C1/C2 of ring A and an acyl (COCH3 and COPh) or phenylsulfonyl (SO2 Ph and SO2 C6 H4 -3-CH3 ) functionality at the N6 position of ring B of the aporphine scaffold were synthesized and evaluated for their arachidonic acid (AA)-induced antiplatelet aggregation inhibitory activity and 2,2-diphenyl-1-picrylhydrazyl (DPPH) free-radical-scavenging antioxidant activity, with acetylsalicylic acid and ascorbic acid as standard references, respectively. The preliminary structure-activity relationship related to AA-induced platelet aggregation inhibitory activity results showed that the aporphine analogues 1-[1,2,9,10-tetramethoxy-6a,7-dihydro-4H-dibenzo[de,g]quinolin-6(5H)-yl]ethanone and 1-[2-(benzyloxy)-1,9,10-trimethoxy-6a,7-dihydro-4H-dibenzo[de,g]quinolin-6(5H)-yl]ethanone to be the best compounds of the series. Moreover, the DPPH free-radical-scavenging antioxidant activity results demonstrated that the aporphine analogues 1,2,9,10-tetramethoxy-6-(methylsulfonyl)-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline, 2-ethoxy-1,9,10-trimethoxy-6-(methylsulfonyl)-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline, 1-ethoxy-2,9,10-trimethoxy-6-(methylsulfonyl)-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline, 2,9,10-trimethoxy-6-(methylsulfonyl)-1-propoxy-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline, and 1-(benzyloxy)-2,9,10-trimethoxy-6-(methylsulfonyl)-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline were the best compounds of the series. Moreover, in silico molecular docking simulation studies of the active analogues were also performed.

Molecular characterization of anti-platelet aggregating proteins in salivary gland extracts of Hyalomma anatolicum ticks

Tick saliva is a cocktail of potent anti-haemostatic, anti-inflammatory and immunomodulatory molecules that help the ticks to obtain a blood meal from their vertebrate hosts. Thrombin is considered as an activator for stimulation of blood platelets at different receptor sites and thrombin induced platelet aggregation depends on the interaction among thrombin exosites and the receptors on platelet membrane. Thus, the present study was planned to isolate and characterize the salivary anti-platelet aggregating proteins from Hyalomma anatolicum ticks. A hundred pairs of salivary glands were dissected out and extract was prepared by fractionation of proteins by gel filtration chromatography. The fractions so obtained were tested for thrombin induced platelet aggregation and platelet-collagen adhesion inhibition activities. Proteins in fraction nos. 28, 29, 30, 31, 32, 42, 58, 111 and 112 were found to inhibit platelet aggregation. These proteins also showed significant inhibitory effects on platelet adhesion to collagen when compared to agonist thrombin. This indirectly indicates inhibition of glycoprotein 1b (GP1b), glycoprotein VI (GPVI) and integrin a2b1 receptors of platelets as these proteins are involved in aggregation of platelets for formation of platelet plug. On electrophoretic separation of fractions by SDS-PAGE, the proteins/peptides present in initial fractions were having approximate molecular weight in the range of 6.5 to 113.4kDa. However, in the fraction no. 112 only single prominent protein band of approximate 65.4kDa was present and is expected to inhibit platelet aggregation possibly through inhibition of platelet receptors such as proteins platelet activating receptors-1 and 4 playing direct role in platelet aggregation. In conclusion, there are possibilities of exploring these anti-platelet aggregating proteins/peptides for therapeutics against cardio-vascular disorders as well as for raising anti-tick vaccine.